1. Field of the Invention
The present invention relates to blockade of the 12(S)-HETE cell surface receptor as a treatment for conditions of the body which result from stimulation or overstimulation of the receptor. 12(S)-HETE, a product of the 12-lipoxygenase pathway, mediates the hyperproliferative and inflammatory responses present in such diseases as atherosclerosis, psoriasis, diabetes, and cancer. 12(S)-HETE also mediates inflammatory responses and cell death in some cell types, particularly pancreatic islet beta cells and nerve cells. Blockade of the 12(S)-HETE receptor ameliorates the symptoms and arrests the mitogenic cellular responses.
2. Background
Lipoxygenases (LO) are metabolic enzymes which catalyze the stereospecific oxygenation of polyunsaturated fatty acids to hydroperoxy fatty acids (Hamberg et al., J. Biol. Chem. 242:5329-5335 (1967)). The physiological function of 12-LO, the mammalian enzyme which catalyzes the oxygenation of arachidonic acid to (S)-12-hydroperoxyeicosatetraenoic acid (12-HPETE) and (S)-12-hydroxyeicosatetraenoic acid (12(S)-HETE), is unclear. 12-LO exists as two isoforms which are the products of different genes, leukocyte-type 12-LO and platelet-type 12-LO, which share 65% homology at the amino acid level (Izumi et al., Proc. Natl. Acad. Sci., USA 87:7477-7481 (1990); Funk et al., Proc. Natl. Acad. Sci. USA 87:5638-5642 (1990)). The products of the 12-LO pathway, such as 12(S)-HETE, have been shown to play important roles in diseases such as atherosclerosis, diabetes, and cancer. 12(S)-HETE has direct mitogenic and hypertrophic effects in vascular cells. It is also a potent chemoattractant for vascular smooth muscle cells (VSMC) and can activate oncogenes such as c-fos and ras and key growth-related kinases such as mitogen-activated protein kinases (ERK, JNK, PAK, p38) and protein kinase C. New results also indicate that 12(S)-HETE can directly increase monocyte binding. Human aortic endothelial cells incubated with 12(S)-HETE for four hours prior to monocyte adhesion assays resulted in an average increase of 3-fold (range of 1.5-5 fold) in monocyte binding as compared to untreated cells. In addition, glucose-induced monocyte adhesion was abrogated by the inhibition of 12-LO using both phenidone, a non-specific LO inhibitor, and baicalein, a more specific 12-LO inhibitor. The adhesion caused by 12-LO products appears to be monocyte-specific.
The 12-LO pathway is activated in pancreatic islets by cytokines and may participate in islet cell destruction. In inflammatory diseases, this pathway plays crucial roles in transmitting distinctive signals within the cell. Using inhibitors of the 12-LO enzyme pathway, researchers have been able to prevent inflammation and cellular damage. Furthermore, VSMC cultured under high glucose (HG) conditions produce increased amounts of 12(S)-HETE (Natarajan et al., Proc. Natl. Acad. Sci. USA 90:4947-4951 (1993). Thus, this pathway may be key to the accelerated cardiovascular disease observed in diabetes.
The LO pathway also plays a role in the growth-promoting effects of angiotensin II (AII) and in the chemotactic effects of platelet-derived growth factor: the products of the 12-LO pathway, and 12(S)-HETE in particular, are associated with the hypertrophic, hyperplastic, and mitogenic effects induced by AII. Wen et al., 271 Am. J. Physiol. (40 Cell Physiol.) C1212-C1220 (1996); (Natarajan et al., Hypertension 23:I142-I147 (1994)). The proliferative effects of AII are inhibited by baicalein, a LO inhibitor. The mitogenic effects of 12(S)-HETE are similar to those of AII and are abrogated by pertussis toxin, implicating a G-protein mechanism. The 12-LO enzyme pathway is known to generate proinflammatory mediators in a variety of cells (O. R. Etingin et al., J. Lipid Res. 31:299-305 (1990); V. A. Folcik and M. K. Cathcart J. Lipid Res. 34:69-79 (1993)). Human and rat pancreatic B-cells specifically express active leukocyte type 12-LO (V. P. Shannon et al., Am. J. Physiol. 263:E828-E836 (1992): D. S. Bleich et al., Endocrinol. 136:5736-5744 (1995)). Recent evidence implicates products of the 12-LO pathway in nerve cell death associated with Parkinson's disease, Alzheimer's disease and other inflammatory nerve cell conditions (Neuron 19:453-463 (1997)).
Because 12(S)-HETE has several biological effects linked to cellular growth in vascular smooth muscle and cardiac fibroblasts (Natarajan et al., Hypertension 23:I142-I147 (1994); Wen et al., Am. J. Physiol. 211:C1212-C1220 (1996)), it is implicated in the etiology of cardiovascular disease. Further evidence that 12(S)-HETE is responsible for the cellular responses seen in cardiovascular disease in diabetic patients includes the fact that monocyte binding to cultured human aortic endothelial cells increases in chronic high glucose conditions, and that this is coincident with increased formation of LO products such as 12(S)-HETE. (Kim et al., Diabetes 43:1103-1107 (1994)). Furthermore, treatment of aortic endothelial cells with 12(S)-HETE increases monocyte binding, likely by stimulating JNK activity and inducing CS-1. 12(S)-HETE can also stimulate vascular endothelial growth factor (VEGF) gene expression in vascular smooth muscle (Am. J. Physiol. 273: H2224-H2231 (1997)). VEGF has been linked to angiogenesis in diabetic retinopathy, tumor growth and atherosclerotic vascular disease. 12(S)-HETE is also regarded as a mediator of inflammation and hyperproliferation of the skin (Arenberger et al., Skin Pharmacol. 6:148-151 (1993); Gross et al., J. Invest. Dermatol. 94:446-451 (1990)) and is therefore implicated in skin diseases. 12(S)-HETE has been shown to enhance tumor cell adhesion to endothelial cells. (Honn et al., Cancer Metastasis Rev. 13:365-396 (1994)). 12(S)-HETE can directly increase p21 activated kinase (PAK). The effect appears to be through activation of small GTP binding proteins such as RAC and through activation of PI3K.
The precise mechanisms of 12(S)-HETE action are not clear, however recent studies have shown that the LO product, 12(S)-HETE, activates c-jun amino terminal kinase (JNK) (Wen et al., Circ. Res. 81:651-655 (1997)). JNK is a small GTP-binding protein and a member of the MAP kinase family which is involved in cellular growth, inflammation, and apoptosis (Force et al., Circ. Res. 78:947-953 (1994)) and in cell cycle progression through G.sub.1 (Olson et al., Science 269:1270-1272 (1995)). Evidence shows that JNK can serve as a positive or negative modulator of cell growth in different cells. Olson et al., 269 Science 1270-1272 (1995); Yan et al., 372 Nature 798-800 (1994). 12(S)-HETE activation of JNK may also be the mediator of cytokine-induced pancreatic B-cell damage (Bleich et al., Biochem. Biophys. Res. Commun. 230:448-451 (1997)).
Newer evidence indicates that the growth factor and potent vasoconstrictor AII, linked to type-1 receptor activation, can activate JNK and PAK (Wen et al., Circ. Res. 81:651-655 (1997); Schmitz et al., Circ. Res. 82:1272-1278 (1998)). Furthermore, AII can modulate serum deprivation-induced apoptosis by increasing JNK activity in vascular smooth muscle cells, Sueror et al., Circulation, Supp. 1, I-281 (1994), mediated by lipids derived from the 12-LO pathway, such as 12(S)-HETE. This indicates that 12-LO products participate in JNK activation at least in part through G.sub.1 -protein signaling. The ability of pertussis toxin to block the activation of JNK by 12(S)-HETE also supports the theory that 12(S)-HETE is a mediator of AII-induced JNK activation through a G.sub.1 -mediated pathway.
While several studies have demonstrated the potent biological effects of lipoxygenase products, the mechanisms of action of these effects are not known. Some reports have hinted at the presence of 12(S)-HETE receptors on transformed cells. Binding sites for 12(S)-HETE have been detected in carcinoma cells (Herbertsson and Hammarstrom, FEBS 298:249-252 (1992), on melanoma cells (Liu et al., Proc. Natl. Acad. Sci. USA 92:9323-9327 (1995), and in a human epidermal cell line (Gross et al., J. Invest. Dermatol. 94:446-451 (1990); Suss et al., Exptl. Cell Res. 191(2):204-208 (1990)).
The 12(S)-HETE receptors described in carcinoma cells are cytosolic receptors (Herbertsson and Hammarstrom, Biochem. Biophys. Acta 1244:191-197 (1995)), activation of which may mediate 12(S)-HETE induced mRNA production of genes coding for the integrin .alpha..sub.IIb .beta..sub.3 (Chang et al., Biochem. Biophys. Res. Comm. 176:108-113 (1991)). The localization of this receptor is different from plasma cell membrane receptors coupled to a G-protein and acting through second messengers. 12(S)-HETE receptors on the cell surface of murine melanoma cells have been described. These receptors stimulate the second messengers diacylglycerol and inositol phosphate.sub.3 via a G-protein mechanism, resulting in protein kinase C.sub.2 activation. (Liu et al., Proc. Natl. Acad. Sci. USA 92:9323-9327 (1995)). The binding of 12(S)-HETE to these receptors was blocked by 13(s)-hydroxyoctadecadienoic acid, a LO metabolite of linoleic acid, ablating the 12(S)-HETE increased adhesion of the cells to fibronectin. These authors suggest 12(S)-HETE may act in a "cytokine" fashion to regulate responses of adjacent tumor cells, endothelial cells, and platelets.
Receptors for 15-HETE have been identified in mast/basophil (PT-18) cells and were shown to possess properties of G-protein-coupled receptors (Vonakis and Vanderhoek, J. Biol. Chem. 267:23625-23631 (1992). Specific binding of 15-HETE to these receptors stimulated 5-LO, and while 12(S)-HETE was found to be an effective competitor of [.sup.3 H]15-HETE binding to PT-18 cells, suggesting that 12(S)-HETE binds to the specific 15-HETE receptor, the binding of 12(S)-HETE did not stimulate the lipoxygenase. Very recent studies have indicated the activation of a cell surface G-protein-coupled 5-HETE receptor in neutrophils (Capadici et al., J. Clin. Invest. 102:165-175 (1998)).
The high affinity 12(S)-HETE-specific receptors in a human epidermal carcinoma cell line were induced by .gamma.-IFN (Gross et al., J. Invest. Dermatol. 94:446-451 (1990)). Saturation binding of 12(S)-HETE to these receptors did not stimulate cell growth, therefore, the function of these receptors in the skin is entirely speculative, and not related to the AII-induced cellular effects mediated by cell surface 12(S)-HETE receptors in fibroblasts overexpressing the AII receptor and potentially in vascular smooth muscle cells. Two recent studies have indicated two additional agents which could reduce 12(S)-HETE binding (Kemeny and Ruzicka, Agents Actions 32:339-342 (1991); Kemeny et al., Arch. Dermatol. Res. 283:333-336 (1991)).
Specific inhibitors of 12-LO have been described. Gorins et al., J. Med. Chem. 39:4871-4878 (1996). In that study, a series of substituted (carboxyalkyl)benzyl ethers were found to be selective inhibitors of leukocyte-type 12-LO. These inhibitors of 12-LO acted by serving as structural analogs for the enzyme. Gorins et al., J. Med. Chem. 39:4871-4878 (1996). The 5-LO inhibitor, 2-phenylmethyl-1-naphthol (DuP654), has also been shown to specifically inhibit binding of 12(S)-HETE to receptors on the human epidermal cell line SCL-II. Arenberger et al., Skin Pharmacol 6:148-151 1993).
In vivo, inhibition of 12-LO has lowered blood pressure in several models of hypertensive animals, including rats (Stern et al., Am. J. Physiol. 257:H434-H443 (1989); Nozawa et al., Am. J. Physiol. 259:H1447-H1780 (1990)). In addition, blockage of 12-LO activity has alleviated the growth-factor induced effects of 12-HETE in vascular cells. This, along with the known increased expression of 12-LO observed in animal models of diabetes (Gu et al., Am. Diabet. Assoc. Meeting (1996); Natarajan et al., Intl. Aldosterone Meeting (1998)) and diabetes induced accelerated atherosclerosis (Gerrity et al., Circulation I175 (1997)) strongly implicate 12-HETE and the 12-LO pathway in the etiology of these diseases. The harmful effects of 12-LO activation are ameliorated by blocking the production of 12(S)-HETE, providing the rationale for a method of treatment which focusses on preventing 12(S)-HETE binding to its receptor.
There is currently no inhibitor of 12(S)-HETE receptor binding in clinical use. Due to the existence of several isoforms of 12-LO, blockage of the 12-HETE receptor is a more specific and direct way to correct a disease state in which there is increased production of 12(S)-HETE or the receptors are up-regulated. This invention therefore, could provide the basis for the development of interventions to reduce cardiovascular disease, diabetes, and cancer.